科研之友
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摘要
Protein glutathionylation is an important protein post-translational modification associated with oxidative stress, in which the thiol groups of cysteine residues react with glutathione and form disulfide bonds. Glutathionylation has been shown to affect protein structure and modulate protein function, and is implicated in the regulation of signaling and metabolic pathways. Glutathionylation of human hemoglobin has been known for decades. However, only glutathionylation on Cys-93 of beta-globin has been characterized. In this study, we used nanoflow liquid chromatography-nanospray ionization multistage mass spectrometry (nanoLC-NSI/MSn) under a data-dependent scan mode to identify sites of glutathionylation in human hemoglobin by accurate mass measurement and by their MS2 and MS3 spectra. After human hemoglobin was incubated with oxidized glutathione, alkylated and trypsin digested, all three cysteine residues, i.e., alpha-Cys-104, beta-Cys-93, and, beta-Cys-112, were found to be glutathionylated. Glutathionylation at these sites was also detected in hemoglobin freshly isolated from human blood samples by the consecutive reaction monitoring at the MS3 scan stage, and the extent of modification of each site was quantified relative to the alkylated parent peptide in the selected reaction monitoring (SRM) chromatograms. The peak area ratio of glutathionylated and alkylated parent peptides under MS3 and MS2-SRM, respectively, represents the relative extent of glutathionylation. The extents of glutathionylation at alpha-Cys-104 and beta-Cys-93 in hemoglobin of 20 smokers were significantly higher than those in 20 nonsmokers with a p value of 0.0008 and %26lt;0.0001, respectively. Moreover, there are statistically significant correlations between the extent of glutathionylation at alpha-Cys-104 and beta-Cys-93 and the number of cigarettes smoked per day as well as smoking index. This assay is highly specific and sensitive as it requires as little as 2 mu g of hemoglobin isolated from one drop of blood. These results suggest that this assay might be feasible in measuring the extent of glutathionylation in hemoglobin as a low-invasive biomarker of oxidative stress. To our knowledge, this is the first report identifying all three cysteine residues being glutathionylated in human hemoglobin and quantifying the extent of glutathionylation at the peptide level using multistage mass spectrometry.
- 出版日期2014-5
